Hepcidins form a new gene family of inducible, liver-expressed, cysteine-rich peptides that have been identified in vertebrate animals, from fish to humans.
Human hepcidin, also known as liver expressed antimicrobial peptide (LEAP-1), was initially purified as a 25 amino acid peptide from urine and plasma ultra-filtrates during screens for proteins/peptides with antimicrobial activity. Through an Advanced Technology Program Grant from the U.S. Department of Commerce to Kent Sea Tech Corp., bass hepcidin was later purified from the gill tissues of hybrid striped bass (Morone chrysops×M. saxatilis, hereinafter HSB) based on its antimicrobial activity against an E. coli strain, and was characterized as a second type of native, bioactive hepcidin peptide found in a vertebrate species. In vivo, bass hepcidin gene expression was shown to be strongly up-regulated following clinical infection with the pathogen Streptococcus iniae. The bass hepcidin peptide was shown to contain 21 amino acids like one of the forms of human hepcidin, including eight cysteines involved in four disulfide bonds, and to be predominantly expressed in the liver in the HSB model. Notably, bass hepcidin was the first hepcidin to be isolated from a non-human vertebrate, the first cysteine-rich anti-microbial peptide (AMP) isolated from fish, and the first demonstration of hepcidin gene expression induced by the bacterial infection of a vertebrate.
In addition to their antimicrobial activities, hepcidins were found to play an essential role in iron homeostasis. Such a role was first suggested in studies using subtractive cloning approaches in mice subjected to dietary iron overload, where hepcidin gene expression was up-regulated under iron-overload conditions, and where disruption of the hepcidin gene led to accumulation of iron in the liver and pancreas, as well as iron depletion in resident macrophages. This pattern closely paralleled the iron distribution pattern seen in cases of hereditary hemochromatosis in humans. In another study, over-expression of hepcidin in transgenic mouse pups induced profound anemia and postpartum mortality. These and other observations led to the hypothesis that elevated levels of hepcidin limit dietary iron uptake in duodenal enterocytes and block the release of iron by macrophages, making hepcidin a key regulator/hormone of iron homeostasis in higher vertebrates. The critical role for hepcidin in human iron regulation has recently been corroborated by the connection of deleterious mutations in the hepcidin gene in several consanguine families with severe juvenile hemochromatosis and with the demonstration of abnormal hepcidin gene expression levels in patients with other genetic variants of this disease.
The association of hepcidin with innate immune response derives from the observation of a robust upregulation of hepcidin gene expression after inflammatory stimuli, such as infections, which induce the acute phase response of the innate immune systems of vertebrates. In bass, experimental infection with the Gram-positive bacterial pathogen, Streptococcus iniae, strongly upregulated hepcidin gene expression within 24 hours post infection, and, in mice, hepcidin gene expression was shown to be upregulated by lipopolysaccharide (LPS), turpentine, Freund's complete adjuvant, and adenoviral infections.
Studies conducted with human primary hepatocytes indicated that hepcidin gene expression responded to the addition of interleukin-6 (IL-6), but not to interleukin-1α (IL-α) or tumor necrosis factor-α (TNF-α). Concordant with this observation, infusion of human volunteers with IL-6 caused the rapid increase of bioactive hepcidin peptide levels in serum and urine, and was paralleled by a decrease in serum iron and transferrin saturation. A strong correlation between hepcidin expression and anemia of inflammation was also found in patients with chronic and inflammatory diseases, including bacterial, fungal, and viral infections. These findings, further corroborated in a mouse model, led to the conclusion that induction of hepcidin during inflammation depends on IL-6, and that the hepcidin-IL-6 axis is responsible for the hypoferremic response and subsequent restriction of iron from blood-borne pathogens.
Evidence of the essential role of hepcidin in iron homeostasis and hypoferremia of inflammation has been primarily gathered from genetic studies in humans and mice, because only two native hepcidin peptides have been purified and the respective genes cloned and characterized to date, one from humans and the other from bass. The structure of mature, bioactive, folded, human hepcidin shows it to be an amphipathic molecule composed of two distorted, anti-parallel β-sheets separated by a hairpin loop containing a vicinal disulfide bond (that is, a disulfide bond between adjacent cysteines) and stabilized by three inter-β-sheet disulfide bonds. The distinctive structure of human hepcidin is due to a disulfide bonding pattern that appears to be highly conserved evolutionarily, and to be required for bioactivity as an iron regulatory molecule and as an antimicrobial compound.
To date, the unique structure of the mature, folded, bioactive hepcidin has severely limited the development and application of sensitive, informative, immunoglobulin antibodies and tools to detect a refolded, synthetic hepcidin and partial, linear amino acid sequences by means of methods adapted from the production of single chain antibodies. These failures suggest that antibodies that recognize discontinuous and conformational epitopes of the mature, correctly folded, bioactive hepcidin molecule of interest are required for the sensitive measurement of the bioactive forms of hepcidin in studies of disease.
The central role of hepcidin and its key functions in iron regulation and in the innate immune response to infection necessitates the invention of novel methods and informative diagnostic tools for the measurement of the mature, bioactive forms of hepcidin in vertebrates, for the regulation of hepcidin production in animals, and for the production of a synthetic hepcidin that has a properly folded tertiary structure as in the native configuration. Further, the production, refolding, purification, and validation of synthetic or recombinant hepcidin peptides, and the development of antibodies specific to the native, bioactive, vertebrate forms, will enable the treatment of human and animal diseases and infections.